Project Summary The aim of this research training plan is to utilize single molecule fluorescence imaging techniques coupled with dye based biosensors incorporating novel environment sensitive dyes to study GTPase signaling during phagocytosis. Phagocytosis is a crucial component of our immune response, whereby apoptotic cells and foreign particles such as bacteria and fungi are consumed and degraded by macrophage, neutrophil, or dendritic cells. This results in the activation of cell-to-cell signaling that adapts the immune system to eliminate a localized threat. The inability to encapsulate certain particles (frustrated phagocytosis), such as asbestos and silica, has been shown to result in accumulation of toxic reactive species that can ultimately lead to lung disease and cancer. From target recognition to successful or unsuccessful encapsulation, phagocytosis involves several distinct steps that each invoke unique and complex signaling pathways to control the necessary cytoskeletal restructuring. The Rho GTPases Cdc42 and Rac1 are necessary for Fc? receptor mediated phagocytosis, but the mechanisms that regulate their transient localization and activation are unclear. We propose to characterize the spatio-temporal dynamics of Cdc42 and Rac1 signaling during frustrated phagocytosis using two single molecule imaging techniques: stochastic optical resolution microscopy (STORM) and single particle tracking photoactived localization microscopy (sptPALM). Because the resolution of these techniques depends on the brightness of the probe, and because we wish to monitor both the localization and conformation of single molecules, we propose to utilize solvent sensitive merocyanine (Mero) dyes to develop dye-based biosensors that will be used in live cell super resolution microscopy. We propose several novel synthetic approaches for improving Mero dyes to enable their use for STORM and sptPALM imaging. Namely, an intramolecular imine reaction will be developed to induce the dyes to reversibly and spontaneously blink, enabling direct, additive-free STORM imaging; and, a simple approach for improving the photostability of Mero (and cyanine) dyes will be developed that utilizes hypervalent iodine electrophilic group transfer reagents to install protective electron withdrawing groups onto the polyene chain of the dyes. We will utilize unnatural amino acid mutagenesis to site-selectively incorporate the optimized Mero dyes onto binding domains that selectively recognize the active states of Cdc42 or Rac1, or directly onto positions of the GTPases where the dyes experience environmental changes due to conformational changes upon activation. These biosensors will be used to study the localization and activation of Cdc42 and Rac1 during IgG recognition and frustrated phagocytosis using STORM and sptPALM, providing data that will be used to develop new models for GTPase activity during this important immune process.